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1.
Antiviral Res ; 212: 105568, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36842536

RESUMO

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 µM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1-4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 µM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors.


Assuntos
Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Humanos , Ensaios de Triagem em Larga Escala , Antivirais/farmacologia , Antivirais/uso terapêutico , RNA Polimerase Dependente de RNA , Rilpivirina/farmacologia , Rilpivirina/uso terapêutico , Febre do Nilo Ocidental/tratamento farmacológico , Replicação Viral
2.
Genes (Basel) ; 12(10)2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34680882

RESUMO

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.


Assuntos
DNA Primase/genética , Primers do DNA/genética , Replicação do DNA/genética , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Enzimas Multifuncionais/genética , Sequência de Aminoácidos/genética , Primers do DNA/síntese química , Didesoxinucleotídeos/genética , Humanos , Nucleotídeos/genética
3.
Nucleic Acids Res ; 49(14): 8199-8213, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34302490

RESUMO

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.


Assuntos
DNA Primase/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Enzimas Multifuncionais/genética , Motivos de Aminoácidos/genética , DNA/biossíntese , Dano ao DNA/genética , Humanos , Proteína FUS de Ligação a RNA/genética
4.
Sci Rep ; 10(1): 9343, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518272

RESUMO

A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.


Assuntos
DNA Primase/genética , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Infecções por HIV/enzimologia , Infecções por HIV/genética , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutação , Tenofovir/toxicidade , Animais , Biocatálise , DNA Primase/química , DNA Primase/deficiência , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/deficiência , Estabilidade Enzimática , Técnicas de Silenciamento de Genes , Humanos , Rim/efeitos dos fármacos , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Enzimas Multifuncionais/química , Enzimas Multifuncionais/deficiência , Multimerização Proteica , Estrutura Quaternária de Proteína
5.
Enzymes ; 45: 289-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627881

RESUMO

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.


Assuntos
DNA Primase/metabolismo , Primers do DNA/biossíntese , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Humanos
6.
DNA Repair (Amst) ; 77: 65-75, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30889508

RESUMO

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg2+ or Mn2+, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp114, Glu116 and Asp280) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn2+. Herein, we evidenced that PrimPol Glu116 contributes to error-prone tolerance of 8oxodG more markedly when both Mg2+ and Mn2+ ions are present. Moreover, Glu116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn2+ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu116 was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.


Assuntos
DNA Primase/química , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Ácido Glutâmico , Manganês/metabolismo , Enzimas Multifuncionais/química , Enzimas Multifuncionais/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Primase/genética , DNA Polimerase Dirigida por DNA/genética , Nucleotídeos de Desoxiadenina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Enzimas Multifuncionais/genética , Mutação Puntual , Multimerização Proteica , Estrutura Quaternária de Proteína
7.
Sci Rep ; 9(1): 1121, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718533

RESUMO

PrimPol is a human primase/polymerase specialized in re-starting stalled forks by repriming beyond lesions such as pyrimidine dimers, and replication-perturbing structures including G-quadruplexes and R-loops. Unlike most conventional primases, PrimPol proficiently discriminates against ribonucleotides (NTPs), being able to start synthesis using deoxynucleotides (dNTPs), yet the structural basis and physiological implications for this discrimination are not understood. In silico analyses based on the three-dimensional structure of human PrimPol and related enzymes enabled us to predict a single residue, Tyr100, as the main effector of sugar discrimination in human PrimPol and a change of Tyr100 to histidine to boost the efficiency of NTP incorporation. We show here that the Y100H mutation profoundly stimulates NTP incorporation by human PrimPol, with an efficiency similar to that for dNTP incorporation during both primase and polymerase reactions in vitro. As expected from the higher cellular concentration of NTPs relative to dNTPs, Y100H expression in mouse embryonic fibroblasts and U2OS osteosarcoma cells caused enhanced resistance to hydroxyurea, which decreases the dNTP pool levels in S-phase. Remarkably, the Y100H PrimPol mutation has been identified in cancer, suggesting that this mutation could be selected to promote survival at early stages of tumorigenesis, which is characterized by depleted dNTP pools.


Assuntos
DNA Primase/química , DNA Primase/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Neoplasias/genética , Mutação Puntual , Animais , Ciclo Celular , Linhagem Celular , Simulação por Computador , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Resistência a Medicamentos , Histidina , Humanos , Hidroxiureia/farmacologia , Camundongos , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Nucleotídeos/metabolismo , Tirosina/genética
8.
Nucleic Acids Res ; 46(8): 4138-4151, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29608762

RESUMO

Human PrimPol is a monomeric enzyme whose DNA primase activity is required to rescue stalled replication forks during nuclear and mitochondrial DNA replication. PrimPol contains an Archeal-Eukaryotic Primases (AEP) core followed by a C-terminal Zn finger-containing domain (ZnFD), that is exclusively required for primer formation and for PrimPol function in vivo. The present study describes the sequential substrate interactions of human PrimPol during primer synthesis, and the relevance of the ZnFD at each individual step. Both the formation of a PrimPol:ssDNA binary complex and the upcoming interaction with the 3'-nucleotide (pre-ternary complex) remained intact when lacking the ZnFD. Conversely, the ZnFD was required for the subsequent binding and selection of the 5'-nucleotide that will become the first nucleotide of the new primer strand. Providing different 5'-site nucleotides, we can conclude that the ZnFD of PrimPol most likely interacts with the γ-phosphate moiety of the 5'-site nucleotide, optimizing formation of the initial dimer. Moreover, the ZnFD also contributes to recognize the cryptic G at the preferred priming sequence 3'GTC5'. Dimer elongation to obtain long DNA primers occurs processively and is facilitated by the 5'-terminal triphosphate, indicating that the ZnFD is also essential in the subsequent translocation/elongation events during DNA primer synthesis.


Assuntos
DNA Primase/química , DNA Primase/metabolismo , Primers do DNA/biossíntese , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/química , Enzimas Multifuncionais/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Manganês , Nucleotídeos/metabolismo , Multimerização Proteica , Moldes Genéticos , Dedos de Zinco
9.
Nucleic Acids Res ; 45(15): 9046-9058, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911121

RESUMO

We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.


Assuntos
Substituição de Aminoácidos , Bioensaio , DNA Primase/genética , DNA Polimerase Dirigida por DNA/genética , Enzimas Multifuncionais/genética , Engenharia de Proteínas/métodos , DNA Polimerase Dirigida por RNA/genética , RNA/genética , Arginina/química , Arginina/metabolismo , Benzotiazóis , Clonagem Molecular , DNA Primase/metabolismo , Primers do DNA/síntese química , Primers do DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Diaminas , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Fluorometria/métodos , Expressão Gênica , Biblioteca Gênica , Humanos , Enzimas Multifuncionais/metabolismo , Mutação , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Compostos Orgânicos/química , Ligação Proteica , Multimerização Proteica , Quinolinas , RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tirosina/química , Tirosina/metabolismo
10.
Sci Rep ; 7(1): 783, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28396594

RESUMO

Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.


Assuntos
DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Proteína de Replicação A/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ativação Enzimática , Humanos , Ligação Proteica , Especificidade por Substrato , Moldes Genéticos
11.
Nat Commun ; 7: 13296, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27897270

RESUMO

Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis.


Assuntos
Genoma Humano , Reação em Cadeia da Polimerase/métodos , Análise de Célula Única , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Variações do Número de Cópias de DNA/genética , DNA Primase/química , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Moldes Genéticos , Thermus thermophilus/enzimologia
12.
Nucleic Acids Res ; 44(10): 4855-70, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27131366

RESUMO

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.


Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Leishmania infantum/enzimologia , Animais , Núcleo Celular/enzimologia , DNA Polimerase Dirigida por DNA/química , Leishmania infantum/citologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Camundongos , Mutagênicos/toxicidade , Estresse Oxidativo , Células RAW 264.7 , DNA Polimerase teta
13.
DNA Repair (Amst) ; 29: 127-38, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25746449

RESUMO

PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to "read" certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed.


Assuntos
DNA Primase/metabolismo , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Cátions , DNA/biossíntese , Dano ao DNA , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Humanos , Manganês/química , Enzimas Multifuncionais/química , Conformação de Ácido Nucleico
14.
Mol Cell ; 52(4): 541-53, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24207056

RESUMO

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.


Assuntos
DNA Primase/fisiologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/fisiologia , Enzimas Multifuncionais/fisiologia , Sequência de Aminoácidos , Animais , Ácido Apurínico/química , Sequência de Bases , Domínio Catalítico , Núcleo Celular/enzimologia , DNA Polimerase II/química , DNA Polimerase gama , DNA Primase/química , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/química , Desoxiadenosinas/química , Desoxirribonucleotídeos/química , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Enzimas Multifuncionais/química
15.
Nat Struct Mol Biol ; 20(12): 1383-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24240614

RESUMO

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy.


Assuntos
DNA Primase/fisiologia , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Enzimas Multifuncionais/fisiologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Primase/química , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Humanos , Enzimas Multifuncionais/química , Enzimas Multifuncionais/metabolismo , RNA Mensageiro/metabolismo , Fase S , Raios Ultravioleta
16.
Nucleic Acids Res ; 40(3): 1366-80, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21984415

RESUMO

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.


Assuntos
Proteínas de Bactérias/química , DNA Helicases/química , DNA Primase/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus cereus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA Primase/genética , DNA Primase/metabolismo , DNA Primase/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína
17.
Virus Res ; 160(1-2): 1-14, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21708194

RESUMO

The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.


Assuntos
Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Recombinação Genética , Replicação Viral , Vírus/enzimologia , Vírus/crescimento & desenvolvimento , Vírus/genética , Vírus/metabolismo
18.
J Microbiol Methods ; 70(3): 389-94, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17604139

RESUMO

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented beta cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne beta site-specific recombinase is transferred into the background. Protein beta catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.


Assuntos
Bacillus subtilis/genética , Genoma Bacteriano , Mutagênese Insercional/genética , Mutagênese Insercional/métodos , Plasmídeos/genética , Recombinação Genética , Transformação Bacteriana/genética
19.
Nucleic Acids Res ; 34(1): 120-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16407330

RESUMO

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein-DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/fisiologia , Reparo do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/fisiologia , Recombinação Genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Sítios de Ligação , Núcleo Celular/ultraestrutura , Dano ao DNA , DNA Bacteriano/química , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/genética , Deleção de Genes
20.
J Mol Biol ; 351(5): 1007-19, 2005 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-16055153

RESUMO

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.


Assuntos
Bacillus subtilis/virologia , Bacteriófagos/metabolismo , Replicação do DNA , Recombinação Genética , Proteínas não Estruturais Virais/fisiologia , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Dimerização , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Exonucleases/metabolismo , Genoma , Substâncias Macromoleculares , Magnésio/química , Modelos Genéticos , Peso Molecular , Oligonucleotídeos/química , Fosfatos/metabolismo , Mapeamento Físico do Cromossomo , Plasmídeos/metabolismo , Origem de Replicação/genética , Fatores de Tempo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
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